Project Summary. Animal microbiotas are increasingly recognized as essential for host health. The gut microbiota is the richest, and was shown to contribute to diverse host functions. Perturbations in microbiota composition are associated with human disease, raising interest in manipulating the microbiota to promote healthier living or treat pathology. However, current understanding of the factors that shape microbiota composition is still lacking. Studies in vertebrates characterized the effects of diet on microbiota composition, but much less is known about the role of genetic factors, due to high inter-individual variability attributed to genetic heterogeneity. As an alternative, C. elegans enables work with genetically homogenous populations, averaging-out inter-individual variation to discern gene effects. We have established C. elegans as a new model for studying host-microbiota interactions, identifying a reproducible gut microbiota, with commensals that enhance host development and immunity. Our results demonstrate significant contributions of host genetics to shaping of microbiota structure and function and to preferential colonization by beneficial commensals, and identified a role for TGFb signaling in controlling abundance of a beneficial Enterobacter commensal, preventing pathogenic dysbiosis. The goal of the proposed plan is to develop this model to expedite discovery and characterization of host genes that shape microbiota composition and function. Two complimentary experimental pipelines were established in the lab: one takes advantage of composted soil microcosms to grow worms in natural-like environments, using 16S deep sequencing to compare their gut microbiotas to those in their environment, and enabling isolation of environmentally-derived gut commensals; the second, uses synthetic microbiotas consisting of 30 gut isolates, offering tight control over environmental diversity, and interrogated using qPCR with taxa-specific primers. Combining the two approaches, the proposed plan aims to: 1) Characterize TGFb-Enterobacter interactions, determining their specificity, modulation by environmental factors, and the mechanisms underlying their effects on microbiota composition, using it as a case study for detailed investigation of gene-microbiota interactions. 2) Use worm mutants for candidate genes with diverse functions, to characterize gene contributions to microbiota composition and function. 3) Mutagenize worms to identify genes involved in host selectivity toward beneficial commensals; expression of two different fluorescent proteins in two Enterbacter commensals from different Caenorhabditis species revealed preferential colonization of C. elegans by its own beneficial commensal when presented in a mix with a similar C. briggsae commensal; mutants would be sought that do not show this preference. Harnessing the genetic power of C. elegans to studying host-microbiota interactions - establishing the methodologies and commensal collections, will provide essential insights about the factors that shape the gut microbiota. Knowledge gained will form the basis for rational engineering of these communities.